Coatings containing polymer modified enzyme for stable self-cleaning of organic stains

ABSTRACT

Bioactive coatings that are stabilized against inactivation by weathering are provided including a base associated with a chemically modified enzyme capable of enzymatically degrading a component of an organic stain, optionally a lipase or a lysozyme, and optionally a first polyoxyethylene present in the base and independent of the enzyme. The coatings are optionally overlayered onto a substrate to form an active coating facilitating the removal of organic stains or bacterial organic material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/258,564 filed Jan. 26, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/812,087 filed Jul. 29, 2015 (now U.S. Pat. No.10,093,094), which is a continuation of U.S. patent application Ser. No.13/229,277 filed Sep. 9, 2011 (now U.S. Pat. No. 9,121,016), the entirecontents of each which are incorporated herein by reference.

FIELD

The present specification relates generally to coating compositionsincluding active substances and methods of their use to facilitateremoval of organic stains. In specific aspects, the specificationrelates to methods of improving dispersibility of a bioactive enzyme ina polymeric matrix that leads to both improved enzyme stability in thematrix and reduction of weathering.

BACKGROUND

Many outdoor surfaces are subject to stain or insult from naturalsources such as bird droppings, resins, and insect bodies. As a result,the resulting stain often leaves unpleasant marks on the surfacedeteriorating the appearance of the products.

Traditional self-cleaning coatings and surfaces are typically based onwater rolling or sheeting to carry away inorganic materials. These showsome level of effectiveness for removal of inorganic dirt, but are lesseffective for cleaning stains from biological sources, which consist ofvarious types of organic polymers, fats, oils, and proteins each ofwhich can deeply diffuse into the subsurface of coatings. Prior artapproaches aim to reduce the deposition of stains on a surface andfacilitate its removal by capitalizing on the “lotus-effect” wherehydrophobic, oleophobic and super-amphiphobic properties are conferredto the surface by polymeric coatings containing appropriatenanocomposites. An exemplary coating contains fluorine and siliconnanocomposites with good roll off properties and very high water and oilcontact angles. When used on rough surfaces like sandblasted glass,nanocoatings may act as a filler to provide stain resistance. A drawbackof these “passive” technologies is that they are not optimal for use inhigh gloss surfaces because the lotus-effect is based on surfaceroughness.

Photocatalytic coatings are promising for promoting self-cleaning oforganic stains. Upon the irradiation of sun light, a photocatalyst suchas Ti0₂ chemically breaks down organic dirt that is then washed away bythe water sheet formed on the super hydrophilic surface. As an example,the photocatalyst Ti0₂ was used to promote active fingerprintdecomposition of fingerprint stains in U.S. Pat. Appl. Publ.2009/104086. A major drawback to this technology is its limitation touse on inorganic surfaces due to the oxidative impairment of the polymercoating by Ti0₂. Also, this technology is less than optimal forautomotive coatings due to a compatibility issue: Ti0₂ not onlydecomposes dirt, but also oxidizes polymer resins in paint.

Therefore, there is a need for new materials or coatings that canactively promote the removal of organic stains on surfaces or incoatings and minimize the requirement for maintenance cleaning.

SUMMARY

The following summary is provided to facilitate an understanding of someof the innovative features unique to the present specification and isnot intended to be a full description. A full appreciation of thevarious aspects of the specification can be gained by taking the entirespecification, claims, drawings, and abstract as a whole.

A water stabilized active coating material is provided wherein thecoating is capable of degrading a component of an organic stainfollowing immersion of said coating in water for 30 minutes or more,optionally where the coating retains 50% or more activity followingimmersion in water for 30 minutes.

A coating includes a base and an enzyme associated with the base. Anenzyme is optionally chemically modified with one or more polymericmoieties. The coating optionally further includes a firstpolyoxyethylene associated with the base, where the firstpolyoxyethylene is independent of the enzyme; wherein the base, theenzyme, and the first polyoxyethylene form a water-stabilized activecoating composition.

A chemically modified enzyme is optionally a lysozyme or a lipase. Theenzyme is chemically modified by a polymeric moiety, optionally by atleast one molecule of branched polyoxyethylene. The polyoxyethyleneoptionally has a molecular weight between 1,000 and 15,000 Daltons. Insome aspects, the polyoxyethylene further includes a succinimidyl esterprior to reaction with said enzyme. A polymeric moiety is optionallydirectly or indirectly covalently bound to an amino group on the enzymesuch as a terminal amino group or on a lysine. In some aspects apolymeric moiety is directly or indirectly covalently bound to acysteine within the enzyme. In some aspects, a branched polymeric moietyis optionally an eight-arm branched polyoxyethylene.

In some aspects, an enzyme dispersed in base to form a water-stabilizedactive coating composition includes particles of enzyme that contain oneor more enzyme molecules. The average particle diameter is optionallyfrom 1 nanometer to 1 micrometer or any value or range therebetween.

A water-stabilized active coating material optionally is covalentlyattached to at least one component of the base or is non-covalentlyadhered to or admixed into the base. A base is optionally a one or twopart solvent borne system, optionally including a polyurethane.

A process of stabilizing the activity of an enzyme against waterweathering in a coating composition is provided including providing awater-stabilized active coating material is provide that includesassociating one or more polymeric moieties with an enzyme to form achemically modified enzyme and dispersing the chemically modified enzymein a base to form a water-stabilized active coating material. Thedispersing optionally results in protein particles with an averageparticle diameter from 1 nanometer to 1 micrometer, or any value orrange therebetween. A process optionally includes coating a substratewith the active coating material such that the enzyme is capable ofenzymatically degrading a component of an organic stain in contact withthe active coating material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic for forming a water-stabilized active coatingcomposition according to one aspect;

FIG. 2A illustrates large particles formed in a 2K SB coating materialwhen the enzyme is incorporated into the coating material in the absenceof a polymeric moiety as depicted by scanning electron microscopy;

FIG. 2B illustrates large particles formed in a 2K SB coating materialwhen the enzyme is incorporated into the coating material in the absenceof a polymeric moiety as depicted by scanning fluorescence microscopy;

FIG. 3A illustrates effective dispersion of a chemically modified enzymeinto a base according to one aspect using enzyme modified bypolyoxyethylene;

FIG. 3B illustrates effective dispersion of a chemically modified enzymeinto a base according to one aspect using enzyme modified bypolyoxyethylene;

FIG. 3C illustrates effective dispersion of a chemically modified enzymeinto a base according to one aspect using enzyme modified bypolyoxyethylene;

FIG. 3D illustrates effective dispersion of an unchemically modifiedenzyme into a base;

FIG. 4A illustrates a coating formed in the absence of enzyme or apolymeric moiety;

FIG. 4B illustrates the presence of PEG intermixed with enzyme dispersedin the coating;

FIG. 4C illustrates the large particles formed when enzyme (unmodified)is incorporated into a coating;

FIG. 4D illustrates the excellent dispersion of PEGylated enzyme in basematerial (D) demonstrating superior dispersion of enzyme relative to theabsence of a polymeric moiety associated with the enzyme;

FIG. 5 illustrates water-stability of a coating incorporating achemically modified enzyme as measured by residual coating surfaceactivity after water the indicated number of water immersions.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is in no wayintended to limit the scope of the claims, their application, or uses,which may, of course, vary. The specification is described with relationto the non-limiting definitions and terminology included herein. Thesedefinitions and terminology are not designed to function as a limitationon the scope or practice of the claims but are presented forillustrative and descriptive purposes only.

A composition useful as a coating is provided where one or more proteinsassociated with the coating material are optionally chemically modifiedso as to improve coating activity lifetime during and following exposureof a coating to water. The coatings provided herein are bioactivecoatings that have several advantages over other coating materials inthat they present improved lifetime after weathering and easy renewal ofbioactivity by mild abrasion of the coating. Use of coatings containingchemically modified enzymes allows one to regularly renew the bioactivesurface as well as improve other qualities such as shine, protectionfrom the elements, and water runoff.

The coatings as provided herein demonstrate resistance to loss ofactivity due to weathering. Weathering as defined herein includesexposure to water, heat, UV light, or other insult either in theenvironment or in a laboratory. Coatings as provided herein haveunexpected resistance to weathering by exposure to water, such as waterimmersion. As such, the term weathering includes immersion in water.

It is appreciated that the while the description herein is directed tocoatings, the materials described herein may also be substrates orarticles that do not require a coating thereon for promotion of organicstain removal. As such, the word “coating” as used herein means amaterial that is operable for layering on a surface of one or moresubstrates, or may comprise the substrate material itself. In someaspects, a “coating” is exclusive of a substrate such that it is amaterial that may be used to overlay a substrate. As such, the methodsand compositions disclosed herein are generally referred to as an enzymeassociated with a coating for exemplary purposes only. One of ordinaryskill in the art appreciates that the description is equally applicableto substrates themselves.

The provided coatings are based on the catalytic activity of an enzymeto selectively degrade components of organic stains, thus, promotingactive stain removal. Organic stains illustratively include organicpolymers, fats, oils, or proteins. Inventive compositions and processesare provided for the active breakdown of organic stains by awater-stabilized active coating. Coating materials of the prior art havethe capability to degrade organic stains, but the inventors unexpectedlydiscovered that these coatings are rapidly inactivated upon exposure towater such that the expected life of the coating is reduced to the pointof uselessness in the field. Among the nearly infinite possiblemechanisms of promoting stability of coating bioactivity, the inventorsdiscovered that the addition of one or more polymeric moieties on orwith an enzyme prior to incorporation with a base provides fordramatically improved water-stability of the resulting coating material.

A water-stabilized bioactive coating material composition is providedincluding a base with an associated chemically modified enzyme or withan enzyme intermixed with a polymeric moiety, and optionally a firstpolyoxyethylene also associated with the base, where the firstpolyoxyethylene is independent of the enzyme (i.e. not covalently linkedto the enzyme). A composition has utility as a coating for theself-cleaning of organic stains such as food stains, insect stains,fingerprints, and other environmental or artificial insults.

A composition is a water-stabilized coating. The term “water-stabilized”denotes activity of the coating toward the self-cleaning or loosening ofan associated organic stain, where the activity is increased by thepresence of a chemically modified protein relative to the identicalcoating with a non-chemically modified protein. Water-stabilizedoptionally includes coatings that retain 50% to 90%, or any value orrange therebetween, or more activity after coating immersion in waterfor 30 minutes. Water-stabilized optionally includes coatings thatretain 15% or greater activity after coating immersion in water for 90minutes.

In some aspects, a composition is a temporary coating. As used hereinthe term “temporary” is defined as operable for a time between 30minutes and three months. It is appreciated that the outer limit oftemporary is optionally defined by the environmental conditions acoating is subjected to. In some aspects, temporary is at or less thanthree months, optionally, less than 2 months, optionally less than 6, 5,4, 3, 2, or 1 weeks, or any time or range of time therebetween.Optionally, temporary is at or less than 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 day, or any time or range therebetween. In someaspects, the term “temporary” is any time between application of aninventive composition to a substrate and immersion or contact with waterfor 30, 60, or 90 minutes, or more. The reduced enzyme activity afterthe temporary time period is optionally renewed by abrasion of thesurface of the coating to expose previously buried enzyme to thesurface.

A composition includes a base material. As used herein a base materialincludes one or more organic polymeric materials. The combination of oneor more of these materials and an enzyme form a water stabilizedbioactive material (synonymously protein-polymer composite material)that can be used as a substrate material or a coating. Illustrativeexamples of base materials useful for association with one or moreenzymes, optionally chemically modified enzymes, are illustrated in U.S.Patent Application Publication Nos. 2008/0293117 and 2010/0279376.

Preparation of water stabilized bioactive coating materials areillustratively achieved by association, optionally by dissolving,aqueous solutions of enzyme and one or more non-aqueous organicsolvent-borne polymers. Enzyme is optionally dispersed in solvent-borneresin prior to curing. Dispersing of enzyme contrasts with forming largeaverage aggregates (e.g. greater than 5 μm in diameter) of the enzymethat diminish the functionality of the enzymes and enzyme containingbioactive materials. Enzymes are optionally dispersed in the polymericmaterials such that enzymes are unassociated with other bioactiveproteins and/or form relatively small particles by average diameter(e.g. less than 5 μm) of associated proteins. Illustratively, theaverage particle size of enzyme particles in the protein-polymercomposite material is less than 5 μm (average diameter) such as in therange of 1 nm to 5 μm. In some aspects, the average particle size is5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200,100, 50, 25, 20, 15, 10, 5, 1 nm, or less or any value or range at orless than 0.1 nm to 5000 nm. In some aspects, the average particle sizedoes not exceed 5, 4, 3, 2, or 1 μm. Optionally, the average particlesize is 1 μm or less.

Curable protein-polymer compositions are optionally two-componentsolvent-borne (2K SB) compositions. Optionally, one component systems(1K) are similarly operable. Illustratively, an enzyme is entrapped in acoating material such as a latex or enamel paint, varnish, polyurethanegels, or other coating materials. Illustrative examples of incorporatingenzymes into paints are presented in U.S. Pat. No. 5,998,200.

In two-component systems, the two components are optionally mixedshortly before use, for instance, application of the curableprotein-polymer composition to a substrate to form an enzyme containingcoating such as a bioactive clear coat. Generally described, the firstcomponent contains a crosslinkable polymer resin and the secondcomponent contains a crosslinker. Thus, the emulsion is a firstcomponent containing a crosslinkable resin and the crosslinker is asecond component, mixed together to produce the curable protein-polymercomposition.

A polymer resin included in methods and compositions as provided hereincan be any film-forming polymer useful in coating or substratecompositions, illustratively clear coat compositions. Such polymersillustratively include, aminoplasts, melamine formaldehydes, carbamates,polyurethanes, polyacrylates, epoxies, polycarbonates, alkyds, vinyls,polyamides, polyolefins, phenolic resins, polyesters, polysiloxanes; andcombinations of any of these or other polymers.

In some aspects, a polymer resin is crosslinkable. Illustratively, acrosslinkable polymer has a functional group characteristic of acrosslinkable polymer. Examples of such functional groups illustrativelyinclude acetoacetate, acid, amine, carboxyl, epoxy, hydroxyl,isocyanate, silane, vinyl, other operable functional groups, andcombinations thereof.

Examples of organic crosslinkable polymer resins include aminoplasts,melamine formaldehydes, carbamates, polyurethanes, polyacrylates,epoxies, polycarbonates, alkyds, vinyls, polyamides, polyolefins,phenolic resins, polyesters, polysiloxanes, or combinations thereof.

A cross linking agent (crosslinker) is optionally included in thecomposition. The particular crosslinker selected depends on theparticular polymer resin used. Non-limiting examples of crosslinkersinclude compounds having functional groups such as isocyanate functionalgroups, epoxy functional groups, aldehyde functional groups, or acidfunctionality.

In particular aspects of protein-polyurethane composite materials, apolymer resin is a hydroxyl-functional acrylic polymer and thecrosslinker is a polyisocyanate.

A polyisocyanate, optionally a diisocyanate, is a crosslinker reactedwith the hydroxyl-functional acrylic polymer according various aspects.Aliphatic polyisocyanates are optional polyisocyanates used in processesfor making protein-polymer composite materials for clearcoatapplications such as in automotive clearcoat applications. Non-limitingexamples of aliphatic polyisocyanates illustratively include1,4-butylene diisocyanate, 1,4-cyclohexane diisocyanate,1,2-diisocyanatopropane, 1,3-diisocyanatopropane, ethylene diisocyanate,lysine diisocyanate, 1,4-methylene bis (cyclohexyl isocyanate),diphenylmethane 4,4′-diisocyanate, an isocyanurate of diphenylmethane4,4′-diisocyanate, methylenebis-4,4′-isocyanatocyclohexane,1,6-hexamethylene diisocyanate, an isocyanurate of 1,6-hexamethylenediisocyanate, isophorone diisocyanate, an isocyanurate of isophoronediisocyanate, p-phenylene diisocyanate, toluene diisocyanate, anisocyanurate of toluene diisocyanate, triphenylmethane4,4′,4″-triisocyanate, tetramethyl xylene diisocyanate, and meta-xylenediisocyanate.

Curing modalities are those typically used for conventional curablepolymer compositions. Illustratively, curing is achieved by applicationof heat, UV light, or combinations thereof. Optionally, a coatingcomposition is cured by exposure to oxygen or other atmosphere. Someaspects are cured spontaneously without necessary application of othercuring affectors or conditions.

Protein-polymer composite materials are optionally thermosetprotein-polymer composite materials. For example, a substrate or coatingmaterial is optionally cured by thermal curing. A thermal polymerizationinitiator is optionally included in a curable composition. Thermalpolymerization initiators illustratively include free radical initiatorssuch as organic peroxides and azo compounds. Examples of organicperoxide thermal initiators illustratively include benzoyl peroxide,dicumylperoxide, and lauryl peroxide. An exemplary azo compound thermalinitiator is 2,2′-azobisisobutyronitrile.

Conventional curing temperatures and curing times can be used inprocesses according to various aspects. For example, the curing time atspecific temperatures, or under particular curing conditions, isdetermined by the criteria that the cross-linker functional groups arereduced to less than 5% of the total present before curing. Cross-linkerfunctional groups can be quantitatively characterized by FT-IR or othersuitable method. For example, the curing time at specific temperatures,or under particular curing conditions, for a polyurethaneprotein-polymer composite can be determined by the criteria that thecross-linker functional group NCO is reduced to less than 5% of thetotal present before curing. The NCO group can be quantitativelycharacterized by FT-IR. Additional methods for assessing the extent ofcuring for particular resins are well-known in the art. Illustratively,curing may include evaporation of a solvent or by exposure to actinicradiation, such as ultraviolet, electron beam, microwave, visible,infrared, or gamma radiation.

One or more additives are optionally included for modifying theproperties of the protein-polymer composite material and/or theadmixture of organic solvent and polymer resin, the aqueous enzymesolution, the emulsion, and/or the curable composition. Illustrativeexamples of such additives include a UV absorbing agent, a plasticizer,a wetting agent, a preservative, a surfactant, a lubricant, a pigment, afiller, and an additive to increase sag resistance.

A substrate or coating including an enzyme is illustratively anadmixture of a polymer resin, a surfactant and a non-aqueous organicsolvent, mixed to produce an emulsion. The term “surfactant” refers to asurface active agent that reduces the surface tension of a liquid inwhich it is dissolved, or that reduces interfacial tension between twoliquids or between a liquid and a solid.

Surfactants can be of any variety including amphoteric, silicone-based,fluorosurfactants, anionic, cationic and nonionic such as described inK. R. Lange, Surfactants: A Practical Handbook, Hanser GardnerPublications, 1999; and R. M. Hill, Silicone Surfactants, CRC Press,1999, incorporated herein by reference. Examples of anionic surfactantsillustratively include alkyl sulfonates, alkylaryl sulfonates, alkylsulfates, alkyl and alkylaryl disulfonates, sulfonated fatty acids,sulfates of hydroxyalkanols, sulfosuccinic acid esters, sulfates andsulfonates of polyethoxylated alkanols and alkylphenols. Examples ofcationic surfactants include quaternary surfactants and amineoxides.Examples of nonionic surfactants include alkoxylates, alkanolamides,fatty acid esters of sorbitol or manitol, and alkyl glucamides. Examplesof silicone-based surfactants include siloxane polyoxyalkylenecopolymers.

When a bioactive coating is contacted with biological material toproduce a biological stain, the enzyme or combinations of enzymescontact the stain, or components thereof. The contacting allows theenzymatic activity of the enzyme to interact with and enzymaticallyalter the components of the stain improving its removal from thesubstrate or coating.

A composition includes at least one active protein that serves toproduce the bioactive coating. An active protein is a macromolecule thathas functional activity such as that of an enzyme illustratively aprotease or hydrolase. A “protein” as defined herein as three or morenatural, synthetic, or derivative amino acids covalently linked by apeptide bond and possessing the activity of an enzyme. Accordingly, theterm “protein” as used herein includes between 3 and about 1000 or moreamino acids or having a molecular weight in the range of about150-350,000 Daltons. A protein is a molecule with a contiguous molecularsequence three amino acids or greater in length, optionally matching thelength of a biologically produced proteinaceous molecule encoded by thegenome of an organism. Examples of proteins include an enzyme, anantibody, a receptor, a transport protein, a structural protein, or acombination thereof. Proteins are capable of specifically interactingwith another substance such as a ligand, drug, substrate, antigen, orhapten. It is appreciated that a protein is chemically modified by theaddition of one or more homo or heteropolymeric moieties as describedherein. The term “analogue” is exclusive of chemical modification with ahomo or heteropolymeric group with the exception of biotinylation.

A protein is optionally modified from a naked polypeptide sequence suchas by the addition or subtraction of one or more molecules ofphosphorus, sulfur, or by the addition of a pendent group such as abiotin, avidin, fluorophore, lumiphore, or other pendent group suitablefor purification, detection, or altering solubility or othercharacteristic of a protein.

The description herein is directed to a protein that is an enzyme, butit is appreciated that other protein active components are similarlyoperable herein. An enzyme is optionally a bioactive enzyme. A bioactiveenzyme is capable of cleaving a chemical bond in a molecule that isfound in a biological organism, the environment, or in food. A coatingthat is bioactive contains one or more bioactive enzymes. An enzyme isoptionally a protease that is capable of cleaving a peptide bondillustratively including a bacterial protease, or analogue thereof. Aprotein that functions as an enzyme is optionally identical to thewild-type amino acid sequence encoded by a gene, a functional equivalentof such a sequence, or a combination thereof. A protein is referred toas “wild-type” if it has an amino acid sequence that matches thesequence of a protein as found in an organism in nature. It isappreciated that a protein is optionally a functional equivalent to awild-type enzyme, which includes a sequence and/or a structural analogueof a wild-type protein's sequence and/or structure and functions as anenzyme. The functional equivalent enzyme may possess similar or the sameenzymatic properties as a wild-type enzyme, such as catalyzing chemicalreactions of the wild-type enzyme's EC classification, and/or maypossess other enzymatic properties, such as catalyzing the chemicalreactions of an enzyme related to the wild-type enzyme by sequenceand/or structure. An enzyme encompasses its functional equivalents thatcatalyze the reaction catalyzed by the wild-type form of the enzyme(e.g., the reaction used for EC Classification). As an illustrativenon-limiting example, the term “amylase” encompasses any functionalequivalent of an amylase that retains amylase activity though theactivity may be altered such as by increased reaction rates, decreasedreaction rates, altered substrate preference, increased or decreasedsubstrate binding affinity, etc. Examples of functional equivalentsinclude mutations to a wild-type enzyme sequence, such as a sequencetruncation, an amino acid substitution, an amino acid modification,and/or a fusion protein, etc., wherein the altered sequence functions asan enzyme.

An enzyme is immobilized into or on coatings and catalyzes thedegradation of organic stain components into smaller molecules. Withoutbeing limited to one particular theory, the smaller product moleculesare less strongly adherent to a surface or coating such that gravity orgentle rinsing such as with water, air, or other fluid promotes removalof the organic stain material from the coating. Thus, the providedaspects have utility as a composition and method for the active removalof organic stains from surfaces.

Enzymes are generally described according to standardized nomenclatureas Enzyme Commission (EC) numbers. Examples of enzymes operable hereininclude: EC1, oxidoreductases; EC2, transferases; EC3, hydrolases; EC4,lyases; EC5, isomerases; or EC6, ligases. Enzymes in any of thesecategories can be included in a composition according to variousaspects.

In some aspects, an included enzyme is a hydrolase such as aglucosidase, a protease, or a lipase. Non-limiting examples ofglucosidases include amylases, chitinase, and lysozyme. Non-limitingexamples of proteases include trypsin, chymotrypsin, thermolysin,subtilisin, papain, elastase, and plasminogen. Non-limiting examples oflipases include pancreatic lipase and lipoprotein lipase. Illustrativeexamples of proteins that function as enzymes are included in U.S.Patent Application Publication No: 2010/0210745.

Amylase is an enzyme present in some aspects of a coating composition.Amylases have activity that break down starch. Several types of amylasesare operable herein illustratively including α-amylase (EC 3.2.1.1)responsible for endohydrolysis of (1->4)-alpha-D-glucosidic linkages inoligosaccharides and polysaccharides. α-Amylase is illustrativelyderived from Bacillus subtilis and has the sequence found at GenbankAccession No: ACM91731 (SEQ ID NO: 1), or an analogue thereof andencoded by the nucleotide sequence of SEQ ID NO: 2. A specific exampleis α-amylase from Bacillus subtilis available from Sigma-Aldrich Co.,St. Louis, Mo. Additional α-amylases include those derived fromGeobacillus stearothermophilus (Accession No: AAA22227), Aspergillusoryzae (Accession No: CAA31220), Homo sapiens (Accession No: BAA14130),Bacillus amyloliquefaciens (Accession No: ADE44086), Bacilluslicheniformis (Accession No: CAA01355), or other organism, or analoguesthereof. It is appreciated that β-amylases, γ-amylases, or analoguesthereof from a variety of organisms are similarly operable in aprotein-polymer composition.

Specific examples of amylase enzymes illustratively have 1000 U/gprotease activity or more wherein one (1) U (unit) is defined as theamount of enzyme that will liberate the non-protein digestion productform potato starch of Zulkowsky (e.g. starch, treated with glycerol at190° C.; Ber. Deutsch. Chem. Ges, 1880; 13:1395). Illustratively, theamylase has activity anywhere at or between 1,000 U/g to 500,000 U/g, orgreater. It is appreciated that lower activities are operable.

A protease is optionally a bacterial metalloprotease such as a member ofthe M4 family of bacterial thermolysin-like proteases of whichthermolysin is the prototype protease (EC 3.4.24.27) or analoguesthereof. A protease is optionally the bacterial neutralthermolysin-like-protease (TLP) derived from Bacillus stearothermophilus(Bacillus thermoproteolyticus Var. Rokko) (illustratively sold under thetrade name “THERMOASE C160” available from Amano Enzyme U.S.A., Co.(Elgin, Ill.)) or analogues thereof. A protease is optionally anyprotease presented in de Kreig, et al., J Biol Chem, 2000;275(40):31115-20. Illustrative examples of a protease include thethermolysin-like-proteases from Bacillis cereus (Accession No. P05806),Lactobacillis sp. (Accession No. Q48857), Bacillis megaterium (AccessionNo. Q00891), Bacillis sp. (Accession No. Q59223), Alicyclobacillisacidocaldarious (Accession No. Q43880), Bacillis caldolyticus (AccessionNO. P23384), Bacillis thermoproteolyticus (Accession No. P00800),Bacillus stearothermophilus (Accession No. P43133), Bacillus subtilis(Accession No. P06142), Bacillus amyloliquefaciens (Accession No.P06832), Lysteria monocytogenes (Accession No: P34025; P23224), amongothers known in the art.

A wild-type protease is a protease that has an amino acid sequenceidentical to that found in an organism in nature. An illustrativeexample of a wild-type protease is that found at GenBank Accession No.P06874 and SEQ ID NO: 3, with the nucleotide sequence encoding SEQ IDNO: 3 found in Takagi, M., et al., J Bacteriol., 1985; 163(3):824-831and SEQ ID NO: 4.

Methods of screening for protease activity are known and standard in theart. Illustratively, screening for protease activity in a proteaseprotein or analogue thereof illustratively includes contacting aprotease or analogue thereof with a natural or synthetic substrate of aprotease and measuring the enzymatic cleavage of the substrate.Illustrative substrates for this purpose include casein of which iscleaved by a protease to liberate folin-positive amino acids andpeptides (calculated as tyrosine) that are readily measured bytechniques known in the art. The synthetic substrate furylacryloylatedtripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtainedfrom Bachem A G, Bubendorf, Switzerland is similarly operable.

Specific examples of proteases illustratively have 10,000 Units/gprotease activity or more. In some aspects, a protease is a thermolysinwherein one (1) U (unit) is defined as the amount the enzyme that willliberate the non-proteinous digestion product from milk casein (finalconcentration 0.5%) to give Folin's color equivalent to 1 μmol oftyrosine per minute at the reaction initial reaction stage when areaction is performed at 37° C. and pH 7.2. Illustratively, the proteaseactivity is anywhere between 10,000 PU/g to 1,500,000 U/g inclusive orgreater. It is appreciated that lower protease activities are operable.Protease activity is optionally in excess of 300,000 U/g. Optionally,protease activity is between 300,000 U/g and 2,000,000 U/g or higher.

A protein is optionally a lipase. A wild-type lipase is a lipase thathas an amino acid sequence identical to that found in an organism innature. An illustrative example of a wild-type lipase is that found atGenBank Accession No. ACL68189 and SEQ ID NO: 5. An exemplary nucleotidesequence encoding a wild-type lipase is found at Accession No. FJ536288and SEQ ID NO: 6.

Lipase activity is illustratively defined in Units/gram. 1 Unitillustratively corresponds to the amount of enzyme that hydrolyzes 1μmol acetic acid per minute at pH 7.4 and 40° C. using the substratetriacetin (Sigma-Aldrich, St. Louis, Mo., Product No. 90240). The lipaseof SEQ ID NO: 5 may have an activity of 200 Units/gram.

Methods of screening for lipase activity are known and standard in theart. Illustratively, screening for lipase activity in a lipase proteinor analogue thereof illustratively includes contacting a lipase oranalogue thereof with a natural or synthetic substrate of a lipase andmeasuring the enzymatic cleavage of the substrate. Illustrativesubstrates for this purpose include tributyrin and triacetin both ofwhich are cleaved by a triacylglycerol lipase to liberate butyric acidor acetic acid, respectively, that is readily measured by techniquesknown in the art.

A protein optionally functions with one or more cofactor ions orproteins. A cofactor ion is illustratively a zinc, cobalt, or calcium.

Cloning, expressing, and purifying any protein operable herein isachievable by methods ordinarily practiced in the art illustratively bymethods disclosed in: Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992(with periodic updates); and Short Protocols in Molecular Biology, ed.Ausubel et al., 52 ed., Wiley-Interscience, New York, 2002.

Naturally derived amino acids present in a protein illustrativelyinclude the common amino acids alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, and tyrosine. It is appreciated that lesscommon derivatives of amino acids that are either found in nature orchemically altered are optionally present in a protein as well such asalpha-asparagine, 2-aminobutanoic acid or 2-aminobutyric acid,4-aminobutyric acid, 2-aminocapric acid (2-aminodecanoic acid),6-aminocaproic acid, alpha-glutamine, 2-aminoheptanoic acid,6-aminohexanoic acid, alpha-aminoisobutyric acid (2-aminoalanine),3-aminoisobutyric acid, beta-alanine, allo-hydroxylysine,allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylornithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxyisoquinoline, isovaline, 2-hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine.

A protein is obtained by any of various methods known in the artillustratively including isolation from a cell or organism, chemicalsynthesis, expression of a nucleic acid sequence, and partial hydrolysisof proteins. Chemical methods of protein synthesis are known in the artand include solid phase peptide synthesis and solution phase peptidesynthesis or by the method of Hackeng, T M, et al., Proc Natl Acad SciUSA, 1997; 94(15):7845-50. A protein may be a naturally occurring ornon-naturally occurring protein. The term “naturally occurring” refersto a protein endogenous to a cell, tissue or organism and includesallelic variations. A non-naturally occurring protein is synthetic orproduced apart from its naturally associated organism or is modified andis not found in an unmodified cell, tissue or organism.

Modifications and changes can be made in the structure of a protein andstill obtain a molecule having similar characteristics as an activeenzyme (e.g., a conservative amino acid substitution). For example,certain amino acids can be substituted for other amino acids in asequence without appreciable loss of activity or optionally to reduce orincrease the activity of an unmodified protein. Because it is theinteractive capacity and nature of a protein that defines that protein'sfunctional activity, certain amino acid sequence substitutions can bemade in a protein sequence and nevertheless obtain a protein with likeor other desired properties. Proteins with an amino acid sequence thatis not 100% identical to that found in nature are termed analogues. Ananalogue optionally includes one or more amino acid substitutions,modifications, deletions, additions, or other change recognized in theart with the proviso that any such change produces a protein with thesame type of activity (e.g. hydrolase) as the wild-type sequence. Inmaking such changes, the hydropathic index, or the hydrophilicity ofamino acids can be considered. In such changes, the substitution usingamino acids whose hydropathic indices or hydrophilicity values arewithin ±2, those within ±1, and those within ±0.5 are optionally used.

Amino acid substitutions are optionally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include(original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),(Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly:Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg),(Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe),and (Val: Ile, Leu). In particular, aspects of the proteins can includeanalogues having about 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequenceidentity to a wild-type protein.

It is further appreciated that the above characteristics are optionallytaken into account when producing a protein with reduced or increasedenzymatic activity. Illustratively, substitutions in a substrate bindingsite, exosite, cofactor binding site, catalytic site, or other site in aprotein may alter the activity of the enzyme toward a substrate. Inconsidering such substitutions the sequences of other known naturallyoccurring or non-naturally occurring like enzymes may be taken intoaccount. Illustratively, a corresponding mutation to that of Asp213 inthermolysin is operable such as that done by Miki, Y, et al., Journal ofMolecular Catalysis B: Enzymatic, 1996; 1:191-199. Optionally, asubstitution in thermolysin of L144 such as to serine alone or alongwith substitutions of G8C/N60C/S65P are operable to increase thecatalytic efficiency by 5-10 fold over the wild-type enzyme. Yasukawa,K, and Inouye, K, Biochimica et Biophysica Acta (BBA)—Proteins &Proteomics, 2007; 1774:1281-1288. The mutations in the bacterial neutralprotease from Bacillus stearothermophilus of N116D, Q119R, D150E, andQ225R as well as other mutations similarly increase catalytic activity.De Kreig, A, et al., J. Biol. Chem., 2002; 277:15432-15438. De Kreigalso teach several substitutions including multiple substitutions thateither increase or decrease the catalytic activity of the protein. Id.and De Kreig, Eur J Biochem, 2001; 268(18):4985-4991. Othersubstitutions at these or other sites optionally similarly affectenzymatic activity. It is within the level of skill in the art androutine practice to undertake site directed mutagenesis and screen forsubsequent protein activity such as by the methods of De Kreig, Eur JBiochem, 2001; 268(18):4985-4991.

A protein is optionally an analogue of a wild-type protein. An analogueof a protein has an amino acid sequence that when placed in similarconditions to a wild-type protein possess some level of the activity ofa wild-type enzyme toward the same substrate. An analogue optionally has500%, 250%, 200%, 150%, 110%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 25%, 10%, 5%, or any value orrange of values therebetween, the activity of a wild-type protein. Anymodification to a wild-type protein may be used to generate an analogue.Illustratively, amino acid substitutions, additions, deletions,cross-linking, removal or addition of disulfide bonds, or othermodification to the sequence or any member of the sequence may be usedto generate an analogue. An analogue is optionally a fusion protein thatincludes the sequences of two or more wild-type proteins, fragmentsthereof, or sequence analogues thereof.

Methods of screening for protein activity are known and standard in theart. Illustratively, screening for activity of an enzyme illustrativelyincludes contacting an enzyme with a natural or synthetic substrate ofan enzyme and measuring the enzymatic cleavage of the substrate.Illustrative substrates for this purpose include casein, which iscleaved by a protease to liberate folin-positive amino acids andpeptides (calculated as tyrosine) that are readily measured bytechniques known in the art. The synthetic substrate furylacryloylatedtripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtainedfrom Bachem AG, Bubendorf, Switzerland is similarly operable.Illustrative substrates of α-amylase include long chain carbohydratessuch as amylose or amylopectin that make up starch. Other methods ofscreening for α-amylase activity include the colorimetric assay ofFischer and Stein, Biochem. Prep., 1961, 8, 27-33. It is appreciatedthat one of ordinary skill in the art can readily envision methods ofscreening for enzyme activity with the enzyme present in or on a varietyof materials.

A protein is illustratively recombinant. Methods of cloning,synthesizing or otherwise obtaining nucleic acid sequences encoding aprotein are known and standard in the art. Similarly, methods of celltransfection and protein expression are similarly known in the art andare applicable herein. Exemplary cDNA encoding the protein sequence ofSEQ ID NO: 1 is the nucleotide sequence SEQ ID NO: 2. Exemplary cDNAencoding the protein sequence of SEQ ID NO: 3 is the nucleotide sequencefound at accession number M11446 and SEQ ID NO: 4. Exemplary cDNAencoding the protein sequence of SEQ ID NO: 5 is the nucleotide sequenceSEQ ID NO: 6

A protein may be coexpressed with associated tags, modifications, otherproteins such as in a fusion protein, or other modifications orcombinations recognized in the art. Illustrative tags include 6×His,FLAG, biotin, ubiquitin, SUMO, or other tag known in the art. A tag isillustratively cleavable such as by linking to protein via a targetsequence that is cleavable by an enzyme known in the art illustrativelyincluding Factor Xa, thrombin, SUMOstar protein as obtainable fromLifesensors, Inc., Malvern, Pa., or trypsin. It is further appreciatedthat chemical cleavage is similarly operable with an appropriatecleavable linker.

Protein expression is illustratively accomplished followingtranscription of a protein nucleic acid sequence, translation of RNAtranscribed from the protein nucleic acid sequence or analogues thereof.An analog of a nucleic acid sequence is any sequence that whentranslated to protein will produce a wild-type protein or an analogue ofa wild-type protein. Protein expression is optionally performed in acell based system such as in E. coli, Hela cells, or Chinese hamsterovary cells. It is appreciated that cell-free expression systems aresimilarly operable.

It is recognized that numerous analogues of protein are operableincluding amino acid substitutions, alterations, modifications, or otheramino acid changes that increase, decrease, or not do alter the functionof the protein sequence. Several post-translational modifications aresimilarly envisioned as within the scope of the present specificationillustratively including incorporation of a non-naturally occurringamino acid, phosphorylation, glycosylation, addition of pendent groupssuch as biotin, avidin, fluorophores, lumiphores, radioactive groups,antigens, or other molecules.

A protein according to the specification is chemically modified by theaddition of one or more polymeric moieties. Polymeric moietiesoptionally have a molecular weight ranging from 200 to 100,000 Daltons.Polymeric moieties are optionally linear, branched, liable, orcombinations thereof. The polymeric moieties are optionally homomeric orheteromeric. Illustrative examples of polymeric moieties include one ormore molecules of carbohydrate or polyoxyethylene (otherwise known aspolyethylene glycol or “PEG”).

Illustrative examples of polymeric moieties include but are not limitedto: polyalkyl alcohols and glycols (including heteroalkyl with, forexample, oxygen) such as polyoxyethylenes and polyoxyethylenederivatives; dextrans including functionalized dextrans; styrenepolymers; polyethylene and derivatives; polyanions including, but notlimited to, polymers of heparin, polygalacturonic acid, mucin, nucleicacids and their analogs including those with modified ribosephosphatebackbones, polypeptides of glutamate, aspartate, or combinationsthereof, as well as carboxylic acid, phosphoric acid, and sulfonic acidderivatives of synthetic polymers; and polycations, including but notlimited to, synthetic polycations based on acrylamide and 2-acrylamido-2methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similarquarternized polypyridine, diethylaminoethyl polymers and dextranconjugates, polymyxin B sulfate, lipopolyamines, poly(allylamines) suchas the strong polycation poly(dimethyldiallylammonium chloride),polyethyleneimine, polybrene, spermine, spermidine and proteins such asprotamine, the histone polypeptides, polylysine, polyarginine andpolyornithine; and mixtures and derivatives thereof. Suitable additionalpolymers are outlined in Roberts, M. J. et al. (2002) “Chemistry forpeptide and protein PEGylation” Adv. Drug Deliv. Rev. 54, 459-476;Kinstler, O. et al. (2002) “Mono-N-terminal poly(ethyleneglycol)-protein conjugates” Adv. Drug Deliv. Rev. 54; U.S. ApplicationSer. No. 60/360,722; U.S. Pat. Nos. 5,795,569; 5,766,581; EP 01064951;U.S. Pat. No. 6,340,742; WO 00176640; WO 002017; EP0822199A2; WO0249673A2; U.S. Pat. Nos. 4,002,531; 5,183,550; 5,985,263; 5,990,237;6,461,802; 6,495,659; 6,448,369; 6,437,025; 5,900,461; 6,413,507;5,446,090; 5,672,662; 6,214,966; 6,258,351; 5,932,462; 5,919,455;6,113,906; 5,985,236; WO 9428024A1; U.S. Pat. Nos. 6,340,742; 6,420,339;and WO 0187925A2.

Polyoxyethylene includes the generic structure —(CH₂CH₂O)_(n)—, where nis an integer optionally from 2 to 2000. Optionally, n is an integerranging from 50 to 500, optionally from 100 to 250, optionally from 150to 250. Polyoxyethylene is optionally provided in a range of sizesattached to proteins using one or more of a variety of chemistries knownin the art. Polyoxyelthylenes are optionally covalently associated withprimary amines (e.g. lysine side chains or the protein N-terminus),thiols (cysteine residues), or histidines. Lysine occurs frequently onthe surface of proteins, so binding of polyoxyethylene at lysine sidechains produces a mix of reaction products. Since the pKa of theN-terminus is significantly different than the pKa of a typical lysineside chain, it is possible to specifically target the N-terminus formodification. Similarly, as most proteins contain very few free cysteineresidues, cysteines (naturally occurring or engineered) may be targetedfor site-specific interactions with polyoxyethylene.

Polyoxyethylene is optionally end capped where one end is end-cappedwith a relatively inactive group such as an alkoxy group, while theother end is a hydroxyl group that may be further modified by linkermoieties. When the term “PEG” is used to describe polyoxyethylene theterm “PEG” may be followed by a number (not being a subscript) thatindicates a PEG moiety with the approximate molecular weight equal thenumber. Hence, “PEG10000” is a PEG moiety having an approximatemolecular weight of 10,000 Daltons. The inventors have found that someaspects including linear PEG10000 are superior to other PEG molecules.In some bases, enzymes that are covalently associated with a branchedPEG, optionally an 8-arm branched PEG, produces superior resistance toweathering by contact or immersion in water.

The term “PEGylation” as used herein denotes modification of a proteinby attachment of one or more PEG moieties via a linker at one or moreamino acids. The polyoxyethylene (PEG) moiety is illustratively attachedby nucleophilic substitution (acylation) on N-terminal α-amino groups oron lysine residue(s) on the gamma-positions, e.g., with PEG-succinimidylesters. Optionally, polyoxyethylene moieties are attached by reductivealkylation—also on amino groups present in the protein usingPEG-aldehyde reagents and a reducing agent, such as sodiumcyanoborohydride. Optionally, polyoxyethylene moieties are attached tothe side chain of an unpaired cysteine residue in a Michael additionreaction using for example PEG maleimide reagents. Polyoxyethylenemoieties bound to a linker are optionally available from JenKemTechnology USA, Allen, Tex. It is appreciated that any PEG moleculetaught in U.S. Application Publication No: 2009/0306337 is operableherein. U.S. Application Publication No: 2009/0306337 also teachesmethods of attaching PEG groups to a protein. PEG is optionally linkedto a protein via an intermediate ester, amide, urethane, or otherlinkage dependent on the choice of PEG substrate and position ofmodification on a protein.

In some aspects, a protein is an analogue of a hydrolase with theinclusion of additional cysteines to provide site specific incorporationsites for polyoxyethylene. In some aspects, lysine or histidine residuesare substituted with alternative amino acids that do not possess atarget primary amine so as to prevent binding of a molecule ofpolyoxyethylene at that site. The choice of amino acid residues such ascysteines, lysines, or histidines to remove depends on the desiredextent of modification. Optionally, simulation computer programs areused to predict whether modification with a polymer will interfere withthe function of the protein as described in U.S. Pat. No. 7,642,340.

Proteins as used herein are optionally mono-substituted i.e. having onlyone polymeric moiety attached to a single amino acid residue in theprotein molecule or to a N-terminal amino acid residue. Alternatively,two, three, four, or more polymeric moieties are present on a singleprotein. In aspects where protein includes more than one polymericmoiety, it optionally has the same moiety attached to each associatedamino acid group or to the N-terminal amino acid residue. However, theindividual polymer groups may also vary from each other in size andlength and differ between locations on the protein.

Reversible binding of one or more polymeric moieties at one or moresites on a protein is optionally used. In these aspects, the polymer iscovalently attached but is liable upon exposure to weathering such asfor example heating, water washing, or simply over time. The liable bondis optionally the bond between the protein and the polymer or within alinker present between a protein and a polymer.

An inventive process and compositions include one or more activechemically modified proteins incorporated into a base to form a coatingmaterial. The protein is optionally non-covalently associated and/orcovalently attached to the base material or is otherwise associatedtherewith such as by bonding to the surface or by intermixing with thebase material during manufacture such as to produce entrapped protein.In some aspects, the protein is covalently attached to the base materialeither by direct covalent interaction between the protein and one ormore components of the base material or by association via a linker.

There are several ways to associate protein with a base in a coating.One of which involves the application of covalent bonds. Specifically,free amine groups of the protein are optionally covalently bound to anactive group of the base. Such active groups include alcohol, thiol,aldehyde, carboxylic acid, anhydride, epoxy, ester, or any combinationthereof. This method of incorporating protein delivers uniqueadvantages. First, the covalent bonds tether the proteins permanently tothe base and thus place them as an integral part of the finalcomposition with much less, if any at all, leakage of the protein.Second, the covalent bonds provide extended enzyme lifetime. Over time,proteins typically lose activity because of the unfolding of theirpolypeptide chains. Chemical bonding such as covalent bondingeffectively restricts such unfolding, and thus improves the proteinlife. The life of a protein is typically determined by comparing theamount of activity reduction of a protein that is free or beingphysically adsorbed with that of a protein covalently-immobilized over aperiod of time.

A protein is optionally associated with a base at a ratio of 1:1 to 1:20(enzyme:base) by weight. Optionally, a protein is associated with a baseat a ratio of 1:2 to 1:15, optionally 1:4 to 1:12 by weight.

Proteins are optionally uniformly dispersed throughout the substratenetwork to create a homogenous protein platform.

Chemical methods of protein attachment to materials will naturally varydepending on the functional groups present in the protein and in thematerial components. Many such methods exist. For example, methods ofattaching proteins (such as enzymes) to other substances are describedin O'Sullivan et al, Methods in Enzymology, 1981; 73:147-166 andErlanger, Methods in Enzymology, 1980; 70:85-104.

Proteins are optionally present in a coating that is layered upon asubstrate wherein the protein is optionally entrapped in the basematerial, admixed therewith, modified and integrated into the basematerial or layered upon a base material.

A water-stabilized coating composition optionally includes one or moreadditives, optionally for modifying the properties of the compositionmaterial. Illustrative examples of such additives include one or morelight stabilizers such as a UV absorber or radical scavengerillustratively including those described in U.S. patent application Ser.No. 13/024,794 or U.S. Pat. No. 5,559,163, a plasticizer, a wettingagent, a preservative, a surfactant, a lubricant, a pigment, a filler,and an additive to increase sag resistance.

An inventive process optionally includes overlayering (coating) asubstrate with a water-stabilized active coating material such that theprotein is capable of enzymatically degrading a component of an organicstain in contact with the active coating material. A substrate is anysurface capable of being coated with an inventive coating. A substrateis optionally flexible or rigid with flexibility relative to that of apolyvinylchloride sheet with a thickness of 10 mm. A substrate has afirst surface and a second surface wherein the first surface and thesecond surface are opposed. A coating is optionally overlayered on asubstrate on a first surface, a second surface, both, or fullyencapsulates a substrate. The coating of a substrate with awater-stabilized active coating material provides a self-cleaningsurface that promotes the removal or loosening of an organic stain whenpresent on the coating.

The identity of a substrate is limited only by its ability to be coatedwith an inventive composition. Illustratively, a substrate is metal,wood, natural or synthetic polymers such as fiberglass or otherplastics, resins, paints, lacquers, stone, leather, other material, orcombinations thereof. A substrate is optionally an automotive body panelor portion thereof. A substrate is optionally a boat hull or portionthereof. A substrate is optionally a wood floor or a coated wood floor.A substrate optionally includes a subcoating such as wood coated with apolyurethane protectant, or a subcoating is a paint, varnish, or otherprotectant commonly found on substrate. A water-stabilized activecoating material optionally contacts the substrate by overlaying thesubcoating material.

Water-stabilized coatings as provided herein provide good adhesion tosubstrates, protection against environmental insults, protection againstcorrosion, and further provide active properties of the protein. Thus,in certain aspects, coatings of water-stabilized active coating materialprovide enzyme activity on a substrate useful in numerous applicationssuch as detection of an analyte which is a substrate for the enzyme or aligand for a receptor, antibody or lectin. In some aspects, coatingsprovide resistance against staining by enzyme digestion of one or morecomponents of stain producing material.

When a water-stabilized composition is contacted with biological, food,or environmental material to produce an organic stain, the enzyme orcombinations of enzymes contact the stain, or components thereof. Thecontacting allows the enzymatic activity of the protein to interact withand enzymatically alter components of the stain improving its removalfrom the substrate or coating.

Proteins are included in compositions as provided herein in amountsranging from 0.1-50, 1-30, 1-20, 1-10, 2-8, 3-6, or other weight percentof the total weight of the material composition.

Enzyme containing coatings have a surface activity generally expressedin Units/cm². Coatings including a thermolysin such as THERMOASE C160(thermolysin from Bacillus stearothermophilus) optionally havefunctional surface activities prior to exposure to water of greater than0.0075 Units/cm². In some aspects, thermolysin surface activity isbetween 0.0075 Units/cm² and 0.05 Units/cm² or any value or rangetherebetween. Optionally, thermolysin surface activity is between 0.0075Units/cm² and 0.1 Units/cm² or any value or range therebetween.Optionally, thermolysin surface activity is between 0.01 Units/cm² and0.05 Units/cm² or any value or range therebetween. In coatingscontaining α-amylase from Bacillis subtilis, typical surface activitiesprior to exposure to water are at or in excess of 0.01 Units/cm². Insome aspects, α-amylase surface activity is between 0.01 Units/cm² and1.5 Units/cm² or any value or range therebetween. Optionally, α-amylasesurface activity is between 0.01 Units/cm² and 2.5 Units/cm² or anyvalue or range therebetween. Optionally, α-amylase surface activity isbetween 0.01 Units/cm² and 1.0 Units/cm² or any value or rangetherebetween. In some aspects, α-amylase surface activity is at orbetween 0.01 Units/cm² and 4.0 Units/cm². It is appreciated that highersurface activities are achievable by increasing the enzymeconcentration, using enzyme with a higher specific activity such as ananalogue of a wild-type enzyme, or by otherwise stabilizing enzymeactivity during association with a base material.

It is appreciated that the inventive processes of facilitating stainremoval will function at any temperature whereby the protein is active.Optionally, the inventive process is performed at 4° C. Optionally, aninventive process is performed at 25° C. Optionally, an inventiveprocess is performed at ambient temperature. It is appreciated that theinventive process is optionally performed from 4° C. to 125° C., or anysingle temperature or range therebetween.

The presence of protein combined with the material of a substrate or acoating on a substrate, optionally, with water or other fluidic rinsingagent, breaks down stains for facilitated removal.

An inventive process includes providing a coating with an enzyme suchthat the enzyme is enzymatically active and capable to cleave orotherwise modify one or more components of an organic stain. Inparticular aspects, an organic stain is based on organic matter such asthat derived from an insect optionally an insect body, a fingerprint,foodstuffs, or from the environment.

An organic stain as defined herein is a stain, mark, or residue leftbehind after an organism, food, or environmental agent contacts acoating. An organic stain is not limited to marks or residue left behindafter a coating is contacted by an insect body. Other sources of organicstains are illustratively: insect wings, legs, or other appendages; birddroppings; food or components of food; fingerprints or residue leftbehind after a coating is contacted by an organism; or other sources oforganic stains such as bacteria or molecules present in water (oraqueous solvent) or soil.

Methods of preparing water-stabilized active coating materialsillustratively include association of aqueous solutions of protein andcommercially available base materials by mixing such as by propellermixing or hand mixing to a uniform or a non-uniform distribution ofchemically modified protein to produce water-stabilized coatingmaterials.

Various aspects are illustrated by the following non-limiting examples.The examples are for illustrative purposes and are not a limitation onany practice. It will be understood that variations and modificationscan be made without departing from the spirit and scope of thespecification.

Example 1

Materials for Production of Water-Stabilized Active Coating Material.

Materials: α-Amylase KLEISTASE SD80 from Bacillus subtilis (EC 3.2.1.1),lipase (lipase A12 (E.C.3.1.1.3) from Aspergillus niger), Protease N,Protease A, Protin SD AY-10, B. sterothermophilus TLP (THERMOASE C160),and THERMOASE GL30 (low activity preparation of B. sterothermophilusTLP) are obtained from AMANO Enzyme Inc. (Nagoya, JAPAN). Bovine serumalbumin (BSA) from bovine serum, starch from potatoes, starch fromwheat, maltose, sodium potassium tartrate, 3,5-dinitrosalicylic acid,Na₂(PO₄), NaCl, K₂(PO₄), casein, trichloroacetic acid, Folin &Ciocalteu's phenol reagent, Na₂(CO₃), sodium acetate, calcium acetate,tyrosine, p-nitrophenyl palmitate, ethanol, iodine, glucose, maltose,maltotriose, maltohexose, dextrin (10 kDa and 40 kDa) are obtained fromSigma Chemical Co., St. Louis, Mo., U.S.A. Aluminum panels and 8-pathwet film applicator are purchased from Paul N. Gardner Company, Inc.(Pompano Beach, Fla.). An Oster blender (600 watts) and light mayonnaiseare obtained from a local supermarket. Freeze-dried crickets areobtained from Fluker Laboratories (Port Allen, La.). Polyethylene glycol(PEG) derivatives with succinimidyl ester of different molecular weightsare obtained from Fishersci (Pittsburgh, Pa.).

Polyacrylate resin Desmophen A870 BA, and the crosslinker hexamethylenediisocyanate (HDI) based polyfunctional aliphatic polyisocyanate resinDesmodur N 3600 are obtained from Bayer Corp. (Pittsburgh, Pa.). Thesurfactant BYK-333 is obtained from BYK-Chemie (Wallingford, Conn.).1-butanol and 1-butyl acetate are obtained from Sigma-Aldrich Co.(Missouri, USA). Aluminum paint testing panels are purchased from Q-LabCo. (Cleveland, USA). All other reagents involved in the experiments areof analytical grade.

Example 2

Preparation of Enzymes.

Lipase, α-amylase, and thermolysin are each prepared by ultrafiltrationfrom raw powder. For α-amylase, a 150 mL solution from raw powder (6.75g) is prepared in DI water. For thermolysin, a 150 mL solution of 1.5 gB. sterothermophilus thermolysin-like-protease (TLP) is prepared in DIwater. For lipase, a 150 mL solution of 1.5 g lipase A12 is prepared inDI water. The insoluble large impurity in raw powder is removed byfiltration over a 200 nm PTFE filter. The obtained solution has aprotein concentration of 20 mg/mL (measured by the Bradford method) andis maintained on ice.

Ultrafiltration is performed using a 150 mL Amicon cell (cooled withice) with a pressure of 55 psi and an ultrafiltration membrane with acut-off of 30 kDa from Millipore (Billerica, Mass.). Ultrafiltration isrepeated 3 times by refilling the cell back to 150 mL of 50 mM PhosphateBuffered Saline (PBS), pH=7.5 after each run. The final remainingpurified protein solution is quantified by the Bradford method anddiluted to the final working concentration used for chemicalmodification and production of coating materials in 50 mM PBS, pH=7.5.Coatings are made using a solution of 50, 100, 200, or 300 mg/mL ofpurified enzyme prior to or following chemical modification with one ormore polymeric moieties.

Example 3

PEGylation of enzyme. Purified enzyme (1 mL, 140 mg/ml) (α-amylase,thermolysin, or lipase) is mixed with PEG (monofunctional linearPEG10000, PEG12000, PEG20000, or 8-arm branched PEG (PEGN-Hydroxysuccinimide ester purchased from NANOCS Inc. (New York, N.Y.),in DMSO) derivatized with succinimidyl ester at a mole ratio of 1:5enzyme:PEG. The 8-arm branched PEG has a molecular weight of 10,000Daltons with a comb-shape structure. Each branch has a molecular weightof −1,200 Daltons. The structure of the 8-arm branched PEG has thefollowing structure:

The reaction mixture is incubated in an ice bath for 4 hours undermagnetic stirring at 800 rpm. Byproducts are removed by ultrafiltration(50K cutoff MW). Enzyme concentration is adjusted to 140 mg/ml forpreparation of coating materials, to 1 mg/ml for SDS-PAGE, and to 0.1mg/ml for activity assays. Some preparations further involve isolationof non-reacted PEG by filtration with a filter with an appropriatemolecular weight cut-off for each PEG used in the PEGylation reactions.

Example 4: Preparation of Coating Materials

Either unmodified (optionally fluorescently labeled) or PEG modified(optionally fluorescently labeled) α-amylase, thermolysin, lipase orcombinations of enzymes are prepared in solution solution (600 μL, 140mg/ml) as in Example 3 and admixed to 2.1 g Desmodur A870 together with500 μL n-butyl acetate and 100 μL surfactant (17% v/v in butyl acetate)under vigorous stirring for 1 min by IKA RW 11 Lab-egg stirrer. Theresulting whitish emulsion is subsequently added to 0.8 g DesmophenN3600 and again vigorously mixed for 1 min.

Test panels are coated with a coating composition by drawn-downapplication of wet film using an 8-path wet film applicator from Paul N.Gardner (Pompano Beach, Fla.) in the predetermined thickness of 20 μmonto aluminum panels or aluminum foil. The resultant coating is flashed10 minutes in air and then cured in oven at 70° C. for 4 hours. Anillustrative process of preparing a water-stabilized bioactive coatingcomposition and applying the composition to a substrate is illustratedin FIG. 1.

Coatings on the substrates are analyses by both fluorescent microscopyand scanning electron microscopy (SEM) to determine the enzymedispersion in the base materials. For SEM characterization,cross-sectionized samples are prepared by coating on the heavy dutyReynolds Wrap® aluminium foil (Richmond, Va.) using an applicator. Thefully cured coatings are torn and the resulting cross-sections of thefractured polymers are sputtered with Au—Pd. The unchemically modified(e.g. not PEGylated) enzymes show large average particle formation inexcess of 5 μm. FIGS. 2A and B. In contrast, the enzymes that aremodified by polyoxyethylene show much reduced average particle sizesindicating dispersion of the enzyme in the base materials. FIG. 3A-C (Dis a non-chemically modified example).

In other aspects, enzyme is intermixed with polyoxyethylene at similarconcentrations to the covalently associated molecules, but withoutcovalent attachment of the enzyme to the polymeric moiety. Theenzyme/polymeric moiety solution is intermixed with base and cured asabove. FIG. 4 illustrates dispersion of the enzyme in the base materialboth when covalently associated with the PEG and when non-covalentlyassociated with the PEG. FIG. 4A illustrates base material intermixedwith PEG alone. FIG. 4B illustrates physical intermixing, butnon-covalently associated enzyme with PEG demonstrating excellentdispersion of the enzyme in the base material. FIG. 4C illustratesunchemically modified enzyme in base material in the absence of apolymeric moiety illustrating the lack of dispersion of enzyme in thebase material. FIG. 4D illustrates covalently associated PEG dispersedin base material demonstrating superior dispersion of enzyme relative tothe absence of a polymeric moiety associated with the enzyme.

Example 5: Water Weathering Durability of Coatings

Coated aluminum panels formed as in Example 4 are cut to test size of1.2 cm×1.9 cm and subjected to weathering by submersion in roomtemperature DI water for 30 minutes with agitation. The test panels areremoved and rinsed with flowing DI water for 20 seconds followed byassay for remaining enzyme activity. The immersion is repeated between 2and 10 times and the remaining enzyme activity assayed.

Test panels coated with α-amylase containing coatings are assayed bydetermination of amydolytic activity by reacting test panels with theα-amylase substrate 1% w/v potato starch in 20 mM sodium phosphatebuffer with 6.7 mM sodium chloride (pH 6.9). The substrate solution (2mL) is added to one rectangular piece of the coated test panel (1.2cm×1.9 cm) and incubated for 3 min at 25° C. The equivalent amount ofreducing sugar produced is determined using a UV-VIS spectrometer (Cary300-Varian Inc., Walnut Creek, Calif., USA) at 540 nm. One unit ofα-amylase activity is defined as 1.0 mg of reducing sugar (calculatedfrom a standard curve previously calibrated against maltose) releasedfrom starch in 3 min at room temperature.

Coatings prepared with thermolysin are assayed for proteolytic surfaceactivity essentially following the method of Folin and Ciocalteau, J.Biol. Chem., 1927; 73: 627-50. Briefly, 1 mL of 2% (w/v) casein insodium phosphate (0.05 M; pH 7.5) buffer solution is used as substratetogether with 200 μl of sodium acetate, 5 mM calcium acetate (10 mM; pH7.5). The substrate solution is pre-incubated in a water bath for 3 minto reach 37° C. The reaction is started by adding one piece of sampleplate coated with B. sterothermophilus TLP based coating (1.2×1.9 cm)followed by shaking for 10 min at 200 rpm at which time the reaction isstopped by adding 1 ml of 110 mM trichloroacetic acid (TCA) solution.The mixture is incubated for 30 min at 37° C. prior to centrifugation.The equivalent of tyrosine in 400 μL of the TCA-soluble fraction isdetermined at 660 nm using 200 μL of 25% (v/v) Folin-Ciocalteau reagentand 1 mL 0.5 M sodium carbonate. One unit of activity is defined as theamount of enzyme hydrolyzing casein to produce absorbance equivalent to1.0 μmol of tyrosine per minute at 37° C. This result is converted toUnits/cm².

An exemplary residual activity depicting the water-stability of coatingpreparations bases on α-amylase is depicted in FIG. 5. The coatingscontaining unmodified α-amylase lose more than 80 percent of the baseactivity after a second wash (black bars). Following a fourth wash, theremaining activity is undetectable. In coating formulations where theenzyme is intermixed (non-covalently attached) with PEG and theintermixed solution is then dispersed into the base (white bars),significant levels of activity remain in the coating relative tocoatings formed in the absence of PEG. Activity remains detectable after10 washes. Covalent attachment of linear PEG molecules onto theα-amylase produces a coating that is highly resistant to activity losswith greater than 30% enzyme activity remaining after three washes(light gray bars) compared to the unmodified enzyme based coatings thatare nearly inactive after three washes. The greatest relative activitylevels are maintained when α-amylase is covalently associated with abranched PEG molecule. FIG. 5 illustrates approximately 40% enzymeactivity remaining after 10 washes (dark gray bars). These datademonstrate that the presence of a polymeric moiety with the enzyme in acoating composition leads to resistance to inactivation by immersion inwater.

Example 6

Preparation of organic stains and application to coated substrate andself-cleaning activity of coating preparations. For preparation ofinsect matter, 60 g of Freeze-dried crickets are chopped into powder bya blender (Oster, 600 watt) for 10 min. The stain solution is preparedby vigorously mixing 2 grams of cricket powder with 6 mL of DI water. Atemplate of uniform spacing is used to apply the stain on the coatingsurface. The cricket stains are dried at 40° C. for 5 min. Each testpanel is placed into a glass dish subjected to rinsing with 200 mL of DIwater under 300 rpm shaking at RT for various times. The time of thestain removal is recorded. In order to reduce random error, the time ofthe first and last drop removed are not included. The average rinsingtime of eight stain spots is averaged for stain removal time. Testpanels coated with PEGylated thermolysin containing coatings provideimproved stain removal by gentle rinsing as compared to panels coatedwith base material alone.

Amylase containing coatings of are placed on the plastic surfaces ofstandard compact disks or aluminum test panels as in Example 4. A 0.3 gsample of light mayonnaise is placed on various sections of the testpanels followed by air dry at ambient conditions for 2 minutes prior tostanding upright. Light mayonnaise includes large macromolecules such asfat and starch that contribute to its high viscosity and thus to thehigh frictional force on the coating surface that prevents gravitydriven sliding of the mayonnaise when the test panel is tiltedvertically. Coatings containing modified α-amylase catalyze thehydrolysis of the emulsifier resulting in tremendously lowered viscosityas a consequence of a phase separation at the stain-coating interface,thus allowing the stain to slide down the test panel when tiltedvertically.

Similarly, aluminum test panels are coated with PEGylated α-amylase, PEGwith no enzyme, or a coating with no enzyme or PEG as a control. Sometest panels are immersed in water between 1 and 5 times as in Example 5prior to contacting with a stain material. A drop of light mayonnaise isthen placed on the panel, the panel is placed in a vertical position andany gravity driven movement of the light mayonnaise spot are monitored.The test panels coated with enzyme free coating or PEG only coatingsshow no movement of the light mayonnaise following tilting to a verticalposition indicating the absence of coating bioactivity. The coating withPEGylated enzyme, however, shows significant self-cleaning asillustrated by lower adherence of the mayonnaise resulting in the spotmoving down the test panel. The self-cleaning aspects of the coatingsare also maintained after each of the water immersions.

The coatings of Example 4 containing PEGylated lipase are used to testremoval of fingerprints from glass or transparent plastic surfaces. Theself-cleaning of fingerprints by PEGylated lipase containingpreparations is tested on glass substrates. Test panels are coated witheither PEGylated lipase containing base materials or control materials(no enzyme) and incubated at room temperature 24 hours. In someexperiments the coated surfaces are immersed in water between 1 and 5times as in Example 5. The test panels are stained with humanfingerprints or facial skin contact. The coated test panels are thenincubated in an oven at 120° C. for 1 to 6 hours. For bettervisualization of any remaining fingerprints, coatings are washed underrunning DI water (50 mL/sec) for 1 minute and dried using air. Prior toheating each coating is subjected to the same level of staining byfingerprints. Following baking, coatings without enzyme show significantresidual staining while coatings containing PEGylated lipase showgreatly reduced stain remaining with the level of residual fingerprintstaining reduced with increased enzyme concentration. The level of stainremoval is also maintained for test panels that were immersed in water.

Various modifications of the present specification, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified or synthesized by one of ordinaryskill in the art without undue experimentation. Methods of proteinproduction and purification are similarly within the level of skill inthe art.

REFERENCE LIST

-   Harris, J. M. and Kozlowski, A. (2001). Improvements in protein    PEGylation: pegylated interferons for treatment of hepatitis C. J.    Control Release 72, 217-224.-   Veronese, F. and Harris, J. M. Eds. (2002). Peptide and protein    PEGylation. Advanced Drug Delivery Review 54(4), 453-609.-   Veronese, F. M.; Pasut, G. (2005), PEGylation, successful approach    to drug delivery, Drug Discovery Today 10 (21): 1451-1458.-   Veronese, F. M.; Harris, J. M. (2002), Introduction and overview of    peptide and protein pegylation, Advanced Drug Delivery Reviews 54    (4): 453-456.-   Damodaran V. B.; Fee C. J. (2010), Protein PEGylation: An overview    of chemistry and process considerations, European Pharmaceutical    Review 15 (1): 18-26.-   Harris, J. M.; Chess, R. B. (2003), Effect of pegylation on    pharmaceuticals. Nature Reviews Drug Discovery 2, 214-221.-   Rodriguez-Martinez J. A., et. al. (2008) Stabilization of    a-Chymotrypsin Upon PEGylation. Correlates With Reduced Structural    Dynamics. Biotechnology and Bioengineering, 101, 1142-1149.-   Li, J.; Kao, W. J. (2003), Synthesis of Polyethylene Glycol (PEG)    Derivatives and PEGylated-Peptide Biopolymer Conjugates.    Biomacromolecules 4, 1055-1067.-   United States Patent Application Publication Number 2010/0279376.-   United States Patent Application Publication Number 2008/0293117.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the specificationpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual patent or publicationwas specifically and individually incorporated herein by reference.

The foregoing description is illustrative of particular aspects ofcompositions or methods, but is not meant to be a limitation upon thepractice thereof. The following claims, including all equivalentsthereof, are intended to define the scope of the invention.

1. A water-stabilized bioactive coating composition comprising: a base;an enzyme associated with said base, said enzyme capable ofenzymatically degrading a component of a bacterial organic stain, saidenzyme selected from the group consisting of a lysozyme or lipase, saidenzyme associated with one or more branched polyoxyethylene to form achemically modified enzyme; and a first polyoxyethylene associated withsaid base, said first polyoxyethylene independent of said enzyme;wherein said base, said chemically modified enzyme, and said firstpolyoxyethylene form a water-stabilized active coating composition. 2.The composition of claim 1, wherein said one or more branchedpolyoxyethylene is covalently attached to said enzyme via anintermediate urethane linkage.
 3. The composition of claim 1, whereinsaid one or more branched polyoxyethylene is an eight-armpolyoxyethylene.
 4. The composition of claim 1, wherein said one or morebranched polyoxyethylene has a molecular weight of 10,000 Daltons to20,000 Daltons.
 5. The composition of claim 1, wherein said one or morebranched polyoxyethylene has a molecular weight of 12,000 Daltons orgreater.
 6. The composition of claim 1, wherein said firstpolyoxyethylene has a molecular weight between 1,000 and 15,000 Daltons.7. The composition of claim 1, wherein said first polyoxyethylene andsaid branched polyoxyethylene have equal polymers of oxyethylene.
 8. Thecomposition of claim 1, wherein said base comprises polyurethane.
 9. Thecomposition of claim 1, wherein said base is formed from a two-componentsolvent-borne composition.
 10. The composition of claim 1, wherein saidenzyme is dispersed in said base as aggregates of said chemicallymodified enzyme.
 11. The composition of claim 10, wherein saidaggregates have an average particle diameter of up to 5 micrometers. 12.The composition of claim 10, wherein said aggregates are formed byadding a solution of said chemically modified enzyme to thetwo-component-solvent borne composition prior to formation of thecontinuous film, and allowing the chemically modified enzyme moleculesto aggregate together to form the aggregates.
 13. The composition ofclaim 1, wherein said composition forms a continuous film.
 14. Awater-stabilized bioactive coating composition comprising: a base; afirst polyoxyethylene associated with said base; and an enzymeassociated with said base, said enzyme capable of enzymaticallydegrading a component of a bacterial organic stain, said enzyme selectedfrom the group consisting of a lysozyme or lipase; wherein said enzymeis associated with one or more molecules of a second polyoxyethylene toform a chemically modified enzyme, the second polyoxyethylene having amolecular weight of 10,000 Daltons or greater; wherein said base, saidchemically modified enzyme, and said first polyoxyethylene form awater-stabilized active coating composition.
 15. The composition ofclaim 14, wherein said first polyoxyethylene has a molecular weightbetween 1,000 and 15,000 Daltons.
 16. The composition of claim 14,wherein said first polyoxyethylene and said second polyoxyethylene haveequal polymers of oxyethylene.
 17. The composition of claim 14, whereinsaid second polyoxyethylene is covalently attached to said enzyme via anintermediate urethane linkage.
 18. The composition of claim 14, whereinsaid second polyoxyethylene has a molecular weight of 10,000 Daltons to20,000 Daltons.
 19. The composition of claim 14, wherein said secondpolyoxyethylene is an eight-arm polyoxyethylene.
 20. A process ofstabilizing the activity of an enzyme against water-weathering in acoating composition comprising: associating one or more polymericmoieties of branched polyoxyethylene with an enzyme, said enzymeselected from the group consisting of a lysozyme or lipase and capableof enzymatically degrading a component of a bacterial organic stain, toform a chemically modified enzyme; and dispersing said chemicallymodified enzyme in a base to form a water-stabilized active coatingmaterial.
 21. The process of claim 20, wherein said dispersing resultsin protein particles with an average particle diameter of less than 5micrometers.
 22. The process of claim 20, further comprising coating asubstrate with said active coating material such that said enzyme iscapable of enzymatically degrading a component of a biological materialin contact with said active coating material.
 23. The process of claim20, wherein said branched polyoxyethylene has a molecular weight between1,000 and 15,000 Daltons.
 24. The process of claim 20, wherein saidpolymeric moiety is an eight-arm polyoxyethylene.
 25. The process ofclaim 20, wherein said base comprises polyurethane.
 26. The process ofclaim 20, wherein said base is formed from a two-component solvent-bornecomposition.
 27. The process of claim 20, wherein said enzyme isdispersed in said base as aggregates of said chemically modified enzyme.28. The process of claim 27, wherein said aggregates have an averageparticle diameter of up to 5 micrometers.
 29. The process of claim 27,wherein said aggregates are formed by adding a solution of saidchemically modified enzyme to the two-component solvent-bornecomposition prior to formation of the continuous film, and allowing thechemically modified enzyme molecules to aggregate together to form theaggregates.
 30. The process of claim 20, wherein said composition formsa continuous film.